PROJECT SUMMARY Tau is a microtubule-stabilizing protein that is abundant in neurons. It is a highly soluble, intrinsically disordered protein (IDP) with little tendency for aggregation under native conditions. However, under several experimental conditions and in a variety of neurodegenerative disorders including Alzheimer’s disease, Tau can spread from cell to cell and aggregates as intra-cellular β-sheet fibrilar deposits. Our laboratories have critical new data concerning the temporal, structural and cell biological details of Tau misfolding and fluid-phase assembly—the basis of this proposal. Our research team consists of a cell biologist, a physical chemist, and a theoretical biophysicist. Working together closely in an iterative manner we intend to determine the pathway from normal Tau to disease-related Tau fibrils. The tools for this analysis include (a) cellular systems capable of addressing in vivo Tau interactions, and indirectly its conformational state based on a variety of molecular probes;; (b) site- directed spin labeling, electron paramagnetic resonance (EPR) line shape analysis and pulsed dipolar EPR to determine conformational signatures of Tau;; and (c) fully atomistic modeling of IDP conformations, their populations and energetics, and coarse-grained simulation of higher-order assemblies of Tau. The conceptual flow of the proposal begins with a remarkable observation from the Han lab: When exposed to sub-stoichiometric amounts of heparin, segments of Tau dramatically extend by a nanometer to solvent-expose the hydrophobic PHF6(*) segment capable of stacking into neat β-sheets. This observation correlates with the appearance of fibrils, and thus we refer to this initiating step as “on pathway” seeding. In vivo, Tau is known to populate a vast conformational landscape controlled by alternative splicing, mutations and post-translational modifications. We propose that the IDP Tau populates an ensemble of different conformations with different aggregation propensities, fibril morphologies and interaction partners, depending on the exact Tau variant. However, the defining and specific conformational signatures within this ensemble are unknown. Determining the conformational signatures of aggregation-prone Tau variants is our core objective, while a missing puzzle piece in connecting Tau conformation to cellular interactions is the existence and nature of aggregation intermediates. In this vein, the Han lab discovered that RNA induces liquid-liquid phase separation of Tau in vitro into protein droplets held together by weak electrostatic forces. At the in vivo cellular level, the Kosik lab discovered Tau- tRNA complexes, thereby adding Tau to the growing list of RNA-binding proteins involved in neurodegeneration, and capable of establishing liquid-liquid phase separation in the cytoplasm. The ...